Alzheimer""s disease (AD) is a common dementing brain disorder of the elderly. The key features of the disease include progressive memory impairment, loss of language and visuospatial skills, and behavior deficits. These changes in cognitive function are the result of degeneration of neurons in the cerebral cortex, hippocampus, basal forebrain, and other regions of the brain. Neuropathological analyses of postmortem Alzheimer""s diseased brains consistently reveal the presence of large numbers of neurofibrillary tangles in degenerated neurons and neuritic plaques in the extracellular space and in the walls of the cerebral microvasculature. The neurofibrillary tangles are composed of bundles of paired helical filaments containing hyperphosphorylated tau protein (Lee, V. M and Trojanowski, J. Q, The disordered Cytoskeleton in Alzheimer""s disease, Curr. Opin. Neurobiol. 2:653 (1992)). The neuritic plaques consist of deposits of proteinaceous material surrounding an amyloid core (Selkoe, D. J., xe2x80x9cNormal and abnormal biology of the xcex2-amyloid precursor proteinxe2x80x9d, Annu. Rev. Neurosci 17:489-517 (1994)).
Evidence suggests that deposition of amyloid-xcex2 peptide (Axcex2) plays a significant role in the etiology of Alzheimer""s disease. A portion of this evidence is based upon studies which have been generated from data with regard to familial Alzheimer""s disease. To date, this aggressive form of Alzheimer""s disease has been shown to be caused by missense mutations in (at least) three genes: the amyloid precursor protein (APP) gene itself (Goate, A. et al., xe2x80x9cSegregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer""s diseasexe2x80x9d, Nature 349:704-706 (1991) and Mullan, M. et al., xe2x80x9cA pathogenic mutation for probable Alzheimer""s disease in the APP gene at the N-terminus of xcex2-amyloidxe2x80x9d, Nature Genet. 1:345-347 (1992)), and two genes termed presenilins 1 and 2 (Sherrington, R. et al., xe2x80x9cCloning of a gene bearing missense mutations in early-onset familial Alzheimer""s diseasexe2x80x9d, Nature 375:754-760 (1995) and Rogaev, E. I. et al., xe2x80x9cFamilial Alzheimer""s disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer""s disease type 3 genexe2x80x9d, Nature 376:775-778 (1995)). The missense mutations in APP are located in the region of the protein where proteolytic cleavage normally occurs (see below), and expression of at least some of these mutants results in increased production of Axcex2 (Citron, M. et al., xe2x80x9cMutation of the xcex2-amyloid precursor protein in familial Alzheimer""s disease increases xcex2-amyloid productionxe2x80x9d, Nature 360:672-674 (1992), Cai, X-D. et al., xe2x80x9cRelease of excess amyloid xcex2 protein from a mutant amyloid xcex2 protein precursorxe2x80x9d, Science 259:514-516 (1993) and Reaume, A. G. et al., xe2x80x9cEnhanced amyloidogenic processing of the beta-amyloid precursor protein in gene-targeted mice bearing the Swedish familial Alzheimer""s disease mutations and a humanized Axcex2 sequencexe2x80x9d, J. Biol. Chem. 271:23380-23388 (1996)). Initial analyses of the structure of the presenilins have not suggested whether or not they might alter production of Axcex2, however, recent data has indicated that these mutations cause an increase in Axcex2 secretion (Martins, R. N. et al., xe2x80x9cHigh levels of amyloid-xcex2 protein from S182 (Glu246) familial Alzheimer""s cellsxe2x80x9d, 7:217-220 (1995) and Scheuner, D. et al., xe2x80x9cSecreted amyloid beta-protein similar to that in the senile plaques of Alzheimer""s disease is increased in vivo by presenilin 1 and 2 and APP mutations linked to familial Alzheimer""s diseasexe2x80x9d, Nature Medicine 2:864-870 (1996)). Thus, increased production of Axcex2 is associated with Alzheimer""s disease. Corroborating evidence has been derived from at least two other sources. The first is that transgenic mice which express altered APP genes exhibit neuritic plaques and age-dependent memory deficits (Games, D. et al., xe2x80x9cAlzheimer-type neuropathology in transgenic mice overexpressing V717F xcex2-amyloid precursor proteinxe2x80x9d, Nature 373:523-525 (1995); Masliah, E. et al., xe2x80x9cComparison of neurodegenerative pathology in transgenic mice overexpressing V717F xcex2-amyloid precursor protein and Alzheimer""s diseasexe2x80x9d, J. Neurosci. 16:5795-5811 (1996); Hsiao, K. et al., xe2x80x9cCorrelative memory deficits, Axcex2 elevation, and amyloid plaques in transgenic micexe2x80x9d, Science 274:99-103 (1996)). The second piece of evidence comes from study of patients suffering from Down""s syndrome, who develop amyloid plaques and other symptoms of Alzheimer""s disease at an early age (Mann, D. M. A. and M. M. Esiri, xe2x80x9cThe pattern of acquisition of plaques and tangles in the brains of patients under 50 years of age with Down""s syndromexe2x80x9d, J. Neurol. Sci. 89:169-179 (1989)). Because the APP gene is found on chromosome 21, it has been hypothesized that the increased gene dosage which results from the extra copy of this chromosome accounts for the early appearance of amyloid plaques (Kang, J. et al., xe2x80x9cThe precursor protein of Alzheimer""s disease amyloid A4 protein resembles a cell-surface receptorxe2x80x9d, Nature 325:733-736 (1987); Tanzi, R. E. et al., xe2x80x9cAmyloid xcex2 protein gene: cDNA, mRNA distribution and genetic linkage near the Alzheimer locusxe2x80x9d, Science 235:880-884 (1987)). Taken together with the evidence derived from cases of familial Alzheimer""s disease, the current data suggests that genetic alterations which result in an increase in Axcex2 production can induce Alzheimer""s disease. Accordingly, since Axcex2 deposition is an early and invariant event in Alzheimer""s disease, it is believed that treatment which reduces production of Axcex2 will be useful in the treatment of this disease.
The principal component of the senile plaque is the 4 kDa xcex2-amyloid peptide (Axcex2). Ranging between 39 and 43 amino acids in length, Axcex2 is formed by endoproteolysis of APP. Alternative splicing generates several different isoforms of APP; in neurons, the predominant isoform is of 695 amino acids in length (APP695). As APP traverses the endoplasmic reticulum (ER) and trans-Golgi network (TGN), it becomes N- and O-glycosylated and tyrosine-sulfated. Mature holoprotein can be catabolized in several compartments to produce both non- and amyloidogenic APP fragments.
APP is expressed and constitutively catabolized in most cells. The dominant catabolic pathway appears to be cleavage of APP within the Axcex2 sequence by an enzyme provisionally termed xcex1-secretase, leading to release of a soluble ectodomain fragment known as APPsxcex1. In contrast to this non-amyloidogenic pathway, APP can also be cleaved by unidentified enzymes known as xcex2- and xcex3-secretase at the N- and C-termini of the Axcex2, respectively, followed by release of Axcex2 into the extracellular space. Several different C-terminal fragments are produced as intermediates in APP catabolic processing; of particular interest is the production of an intracellular an 12 kDa C-terminal fragment (C100) which is produced following xcex2-secretase activity and contains the entire Axcex2 sequence.
Abundant evidence suggests that extracellular accumulation and deposition of A xcex2 is a central event in the etiology of AD. However, recent studies have also proposed that increased intracellular accumulation of Axcex2 or amyloid containing C-terminal fragments may play a role in the pathophysiology of AD. For example, overexpression of APP harboring mutations which cause familial AD results in the increased intracellular accumulation of C100 in neuronal cultures and Axcex242 in HEK 293 cells. Moreover, evidence suggests that intra- and extracellular Axcex2 are formed in distinct cellular pools in hippocampal neurons and that a common feature associated with two types of familial AD mutations in APP (xe2x80x98Swedishxe2x80x99 and xe2x80x98Londonxe2x80x99) is an increased intracellular accumulation of Axcex242. Thus, based on these studies and earlier reports implicating extracellular Axcex2 accumulation in AD pathology, it appears that altered APP catabolism may be involved in disease progression.
Numerous studies have suggested that proteolytic cleavage of APP occurs within acidic compartments of the cell based on the inhibitory actions of agents which are known to disrupt intracellular pH and/or acidic organelles. For example, exposure of cells to the monovalent ionophore, monensin, or high concentrations of ammonium chloride (NH4Cl) been shown to decrease APP proteolytic processing accompanied by concomitant alterations in full-length cellular APP. Similarly, the vacuolar H+-ATPase inhibitor bafilomycin A1 (baf A1) has been reported to produce alterations in APP catabolism which are both cell-type and APP mutation specific. However, the ionophores noted above have been found to be toxic, unacceptably inhibit ATP formation, and alter cellular viability and endosomal and lysosomal function.
Thus, in view of the anticipated benefits of modulating APP catabolism as a treatment for diseases such as AD, compositions and methods for modulating APP catabolism in APP-containing cells which do not substantially alter the viability of those cells, have been desired and are addressed by the present invention.
This invention provides methods and compositions, which are useful in the treatment of amyloidosis and conditions and diseases associated therewith, such as Alzheimer""s Disease (AD). The methods of the invention involve administering to a subject a pharmaceutical composition including one or more agents which modulate APP catabolism and ultimately, amyloid deposition. Accordingly, the methods and compositions of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs. The methods of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis. The methods of the invention are based, at least in part, on modulating catabolism of APP in APP-containing cells through the use of a mobile ionophore, such as carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone, which does not substantially alter the viability of those cells. Therefore, the mobile ionophores disclosed herein can modulate amyloid deposition.
The present invention provides methods for modulating APP catabolism, by administering to APP-containing cells an amount of a mobile ionophore composition effective to modulate APP catabolism without substantially altering the viability of said APP-containing cells. In one preferred embodiment, the modulation includes preventing or inhibiting the amyloid deposition.
The present invention also provides methods for treating a disease state associated with arnyloidosis by administering to a subject having APP-containing cells a pharmaceutically effective amount of a mobile ionophore composition effective to modulate APP catabolism without substantially altering the viability of the APP-containing cells. In one preferred embodiment, the disease state associated with amyloidosis is associated with Alzheimer""s Disease.
The present invention further pertains to packaged pharmaceutical compositions for treating a disease state associated with amyloidosis. The package includes a container for holding an effective amount of a pharmaceutical composition and instructions for using the pharmaceutical composition for treatment of amyloidosis. The pharmaceutical composition includes a mobile ionophore composition effective to modulate APP catabolism in APP-containing cells. In one preferred embodiment, the packaged pharmaceutical composition is for treatment associated with Alzheimer""s Disease.
The present invention further pertains to pharmaceutical compositions for treating amyloidosis, including a therapeutically effective amount of a mobile ionophore composition effective to modulate APP catabolism in APP-containing cells without substantially altering the viability of the APP-containing cells, and a pharmaceutically acceptable vehicle.
The present invention further pertains to methods for modulating amyloid deposition in a subject, including administering to a subject having APP-containing cells an effective amount of a mobile ionophore composition effective to modulate APP catabolism without substantially altering the viability of the APP-containing cells, such that modulation of amyloid deposition occurs.